Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into Multifunctional Sensors

Mitochondrial membranes and their microenvironments directly influence and reflect cellular metabolic states but are difficult to probe on site in live cells. Here, we demonstrate a strategy, showing how the widely used mitochondrial membrane localization fluorophore 10-nonyl acridine orange (NAO) can be transformed into a multifunctional probe of membrane microenvironments by monitoring its blinking kinetics. By transient state (TRAST) studies of NAO in small unilamellar vesicles (SUVs), together with computational simulations, we found that NAO exhibits prominent reversible singlet–triplet state transitions and can act as a light-induced Lewis acid forming a red-emissive doublet radical. The resulting blinking kinetics are highly environment-sensitive, specifically reflecting local membrane oxygen concentrations, redox conditions, membrane charge, fluidity, and lipid compositions. Here, not only cardiolipin concentration but also the cardiolipin acyl chain composition was found to strongly influence the NAO blinking kinetics. The blinking kinetics also reflect hydroxyl ion-dependent transitions to and from the fluorophore doublet radical, closely coupled to the proton-transfer events in the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Following the SUV studies, we show by TRAST imaging that the fluorescence blinking properties of NAO can be imaged in live cells in a spatially resolved manner. Generally, the demonstrated blinking imaging strategy can transform existing fluorophore markers into multiparametric sensors reflecting conditions of large biological relevance, which are difficult to retrieve by other means. This opens additional possibilities for fundamental membrane studies in lipid vesicles and live cells.


Imaging Fluorescence Blinking of a Mitochondrial Localization Probe -Cellular Localization Probes Turned into Multifunctional Sensors
Zhixue (S1) is , assuming all NAO fluorophores are in the singlet (ground) state before onset of excitation at = 0.
For a rectangular excitation pulse, Φ is constant throughout the excitation duration and the matrix is not time dependent. The general solution to Eq S1 is then The dependence of the detected fluorescence at time, t, after onset of excitation is then given by ]( ) (S5) , with denoting the excitation cross section, the fluorescence quantum yield and the detection quantum yield of the emission from the singlet (X=1) and doublet (X=2) state, respectively. For the excitation conditions in our study, 10 ≫ 1 • Φ , 2 • Φ , so that we can assume , with = ( • 2 • 2 )/( • 1 • 1 ) 1 2 representing the relative brightness of the doublet state, • 2 , compared to the singlet state,

S2. Spatial distribution of excitation rates, calculation of average rates
The Gaussian shape of the excitation beam means that the excitation photon flux, Φ ( ̅ ), is a function of position in the sample. As a consequence, a detailed TRAST analysis should include a spatial dependence to both the excitation rates and the resulting electronic state populations. The total fluorescence signal on each pixel of the camera then becomes a convolution of [ ]( ) and the microscope collection efficiency function, ( ̅ ), as shown in Eq. (4). However, simulating the whole 3D sample volume, and computing the projected 2D image on the camera, becomes a costly operation when performed in each iteration of the fitting algorithm. While this procedure is possible, and sometimes required, we found that pre-computing an average observed excitation rate, ̂0 1 , for each pixel or ROI to be analyzed, speeds up the fitting significantly, without appreciable loss of accuracy. The approximate ̂0 1 is computed once, before fitting starts, by weighting 01 ( ̅ ) by brightness and collection efficiency, in the following manner ̂1 ( ̅ ) = 01 ( ̅ )/( 10 + 01 ( ̅ )) represents the population of + * 1 1 at onset of excitation, after equilibration between the singlet states + 0 1 and + * 1 1 S3 and the supernatant was filtered using a 0.2 um spin-filter (Corning, NY, USA) to remove large aggregates.
The Doxyl containing vesicles were made by adding 16-DOXYL stearic acid (Sigma-Aldrich) directly to the final vesicle solution and incubated for 5 min. Before fluorescence detection, 250 nM NAO (Sigma-Aldrich) was added to the final vesicle solution and incubated for 10 min.

S4. Preparation of cells.
HEK293A cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (Life Technology), 1% penicillin-streptomycin. Before TRAST measurements, cells were grown in glass-bottom 8well cell culture plates (Nunc Lab-Tek II Chambered Coverglass) for 1-2 days. Thereafter the cell culture medium was replaced with DPBS buffer (Sigma-Aldrich), and 16-DOXYL stearic acid (Sigma-Aldrich) was added to the buffer. Cells were incubated 15 minutes at 37C, and then NAO was added and incubated for another 15 minutes.

S5. Experimental setup for TRAST measurements.
TRAST measurements were carried out on a home-built TRAST setup ( Figure 1A) based on an inverted epifluorescence microscope (Olympus, IX73). Fluorescence is excited by a 488 nm diode laser (Cobolt, 06-MLD, 200 mW) using an excitation filter (Semrock BrightLine 488/10). The laser beam was modulated by an acousto-optic modulator (AOM; AA Opto Electronics, MQ180-A0,25-VIS). The expanded laser beam was defocused by a convex lens, reflected by a dichroic mirror ((FF506-Di03, Semrock)) and then focused close to the back aperture of the objective (Olympus, UPLSAPO 60x/1.20 W) to produce a wide-field illumination in the sample (beam waist ω0=15µm (1/e 2 radius)). The fluorescence signal was collected by the same objective, passed through the same dichroic mirror, split into two detection channels by a dichroic mirror (T647lpxr, Chroma), then passed though double emission filters ((BrightLine 530/55, Semrock) for the green channel and one emission filter (ET670/50m, Chroma) for the red channel to remove scattered laser light, and was then fed to two separate sCMOS cameras (Hamamatsu ORCA-Flash4.0 v2). The experiments were controlled and synchronized by custom software implemented in Matlab. A digital I/O card (PCI-6602, National Instruments) was used to trigger the camera and generate random excitation pulse trains sent to the AOM driver unit. For experiments with modified oxygen concentrations, a stage incubator system (WP and FC-7, Chamlide, Live Cell Instruments) was used.

S6. TRAST data analysis.
The TRAST data was analyzed using a software implemented in Matlab, as previously described 1 . The recorded TRAST data was first pre-processed by subtraction of the static ambient background, optional binning to either larger pixels or regions of interest (ROIs) within the recorded images, and correction for bleaching. The bleaching correction was based on 10 reference frames, recorded in between the regular frames throughout the measurements (see TRAST spectroscopy above). Moreover, by discarding the data S4 from the first 3-4 excitation pulse trains, a close to steady-state could be established. The overall bleaching was then maximally 5-10 % of the total detected intensity in the vesicle experiments. In the cell measurements, where fluorophores were not replenished by vesicle diffusion, the bleaching was higher, in rare cases as high as 50 %, but a similar steady-state as in the vesicle experiments was nonetheless obtained after 3-4 excitation pulse trains.
In all measurements, TRAST curves were produced by calculating 〈 ( )〉 within a region of interest (ROI) corresponding to a 15 μm radius in the sample plane for both the vesicle and live cell measurements, centered on the excitation beam. In both cases, fitting of photophysical rate parameters was then performed by simulating theoretical TRAST curves using Eqs . For each of the environmental conditions/vesicle samples studied at least three separate TRAST curves were recorded. For each condition of cell sample studied, typically six to eight separate TRAST curves were recorded. Error limits of the determined parameter values were calculated as the standard error of the mean, with one parameter at a time kept as a free parameter in the fittings of the TRAST curves. calculating D1 and D2, the pixel-wise fluorescence intensity values of the images were corrected for static ambient background and photobleaching, following the procedure described above. The resulting fluorescence intensity of each image was then normalized and corrected with an instrument response function, similarly as for the vesicle measurement data. To improve photon statistics and minimize effects of stray photons, images were filtered with a 3x3 pixel median filter. In addition, the median value for each pixel, recorded over the sequence of images within each excitation pulse width range, was computed to give S5 a median normalized intensity value for the corresponding pulse width range. The fluorescence intensity differences were then calculated from these normalized values.

Images of and
The local excitation rate, 01 , was determined for each pixel, as previously described. 30 With knowledge of the total laser power onto the sample, the laser excitation intensity distribution was determined from the image of a fluorophore (Rhodamine 6G) solution, recorded at non-saturating excitation conditions. Each pixel of the reference image was then converted into a local irradiance value and the excitation rates at each location were calculated from the excitation cross-sections 1 and 2. To convert the intensity difference value measured for each pixel to rates, a conversion table was

S11. Computational simulations of the photophysical transitions of NAO
To add evidence and further investigate the prerequisites for NAO to act as a light-induced Lewis acid, computational modelling was performed (see Section S10 for methodological details). First, pKa values were calculated, for NAO as well as for AO, serving as a reference molecule having the same chromophore structure as NAO. For AO, our calculations could reproduce flash photolysis and fluorometry data on this dye 12 , with a higher pKa calculated for the S1 state (pKa=16.4) than for the S0 state (pKa=12.3). For the T1 state however, our calculations predict even stronger basic properties (pKa=17.8) than for the S1 state. The calculated pKa values for S0, S1 and T1 states of the AO dye correlate well with the negative Mulliken charges on the intracyclic (>N) nitrogen atom (-0.35e, -0.38e and -0.51e, respectively). This confirms that the AO dye acts as a photo-base after excitation into the S1 state, but also predicts, in some disagreement with previous experimental findings 12 , that the T1 state of AO has stronger basic properties than the S0 state.
For AO acting as a photo-base, its intra-cyclic nitrogen atom has been identified as the site of the protonation 12 . NAO on the other hand, is typically found in its NAO + cationic form, and its intracyclic nitrogen atom is bound to an n-nonyl group, which is then not available as a site of protonation. In agreement with this, our calculated pKa values for NAO were found to be highly negative: -21.0 (S0), -9.5 (T1) and -10.5 (S1). Similarly, the exocyclic nitrogen atoms (-N(CH3)3 groups) for both the AO and the NAO + dyes also do not demonstrate properties of a base, but rather strong acidity, with calculated pKa values which are negative or close to zero: S0(AO)/(NAO) -4.4/-3.3, S1(AO)/(NAO + ) -4.2/-3.0, T1(AO)/(NAO + ) 0.11/0.14.
To be able to account for the pH dependence for the formation of the red emissive species as seen in the S7 TRAST curves (Figure 2D), we next investigated the prerequisites for NAO to act as a Lewis acid, capable to accept an electron pair from an OHanion. First, we considered different centres of the NAO + cation for attachment of OHanions, based on molecular electrostatic potential (MESP) calculations ( Figure S5A). We then found that in-plane OHinteractions with the aromatic, methyl and nonyl protons of NAO + lead to stable H-bonded complexes [NAO + • OH − ] in the ground S0 state as well as in the excited T1 and S1 states.
However, no evident charge redistribution occurred in such [NAO + • OH − ] complexes, compared with the non-coupled OHand NAO + counterparts. On the other hand, positioning OHover the NAO plane (over the inner pyridine ring where the area of positive MESP is clearly pronounced in the T1 state, but not in the S1 or S0 state, Figure S5A Figure 3.

S12. Fitting of experimental TRAST curves measured in the different emission bands, and under different oxygen concentrations, excitation intensities and pH, to evaluate the photophysical model of
To evaluate the photophysical model, we first fitted the experimental TRAST curves measured in the different emission bands, and under different oxygen concentrations, excitation intensities and pH (Figures   2A-D). Several of the fitted parameter values obtained were then used and kept fixed in the fitting of the subsequent experimental TRAST curves. They are summarized in Table S1. First, the TRAST curves recorded at different pH ( Figure 2D displaying a prominent increase with increasing pH, from 0.0210.0034 s -1 up to 0.500.11 s -1 ( Figure S6), in agreement with the model and with + being promoted by hydroxyl ions. was fitted to (7.2 ± 1.1) × 10 −5 s -1 at pH7.4, accounting for photobleaching at a time scale much slower than the other rates in the model. Next, the TRAST curves measured in different emission bands ( Figure 2A) were fitted, with the rate parameters , , + and − fixed to the fitted values in Figure 2D, fitted globally, and the relative brightness parameter Q fitted individually to each curve. The fitted curves well reproduced the experimental TRAST curves, with Q fitted to 0.26 ± 0.028, 0.32 ± 0.0087 and 0.36 ± 0.0050, the higher the fitted values the more red-shifted the emission filter used, and consistent with a doublet radical with red-shifted emission (Figure 2A). In the fitting of the TRAST curves recorded at different excitation intensities ( Figure   2C), Q, + and − were fixed to the fitted values in Figure 2D, and were fitted globally for all curves, and fitted individually to each curve. The fitted parameters were found to be close to the ones fitted to the TRAST curves in Figure 2D, with and fitted to 15.6 ± 3.0 and 0.40 ± 0.053 s -1 , respectively.
Finally, TRAST curves measured under different oxygenation conditions ( Figure 2B) were fitted, with Q and + fixed to the same values as in Figure 2D, found not to vary significantly. In the fittings, they were at least two orders of magnitude lower than the other fitted rate parameters and did not significantly influence these parameters, and we therefore do not further discuss the fitted values.

S13. Effects of spin labels and redox environment.
In FCS and TRAST measurements addition of paramagnetic spin labels can be clearly observed to enhance the transitions between fluorophore singlet and triplet states, in solution as well as in biological membranes 1,16 . We added the spin label doxyl in different concentrations (0 to 100 M) into solutions containing POPC SUVs labelled with NAO, to see if similar effects could be observed also for this dye. Recorded TRAST curves ( Figure S11A) showed decreased triplet state amplitudes with increasing concentrations of doxyl added, while the doublet state amplitudes remained essentially the same. With , + , and Q fixed to values as determined above, and − were individually fitted and could well reproduce the recorded TRAST curves ( Figure S11A, insets). The rate was found to increase with higher doxyl concentrations, consistent with doxyl acting as a triplet state quencher, thereby also decreasing the overall formation rate of doublet radicals.
In contrast, − was found to decrease upon adding doxyl, which can be attributed to the lower concentrations of hydroxyl radicals then being formed, and to doxyl in addition acting as a radical scavenger.

S9
Since the relative decreases of the overall formation rate and of the − rate were similar, the population of the doublet radical state did not change significantly upon addition of doxyl. The effects of doxyl on the and − of NAO are thus as prominent as those previously found for the deactivation rates of triplet and photo-oxidized states of rhodamine dyes, which can be used for low-frequency collisional interaction or compartmentalization studies in biological membranes and cells 1,16 .
To investigate effects on the transitions from the redox environment, we first added sodium ascorbate (NaAc) in different concentrations (0-2mM) to the same POPC vesicles with NAO. FCS and TRAST measurements have shown that addition of ascorbate can strongly influence dark state transitions of many fluorophores, promoting recovery of photo-oxidized fluorophores and thus enhancing fluorescence emission, but also enhancing photo-reduction 17 . For NAO, we found that when NaAc was added the population of doublet radicals increased, while no effect was noticed on the triplet state kinetics ( Figure S11B). With , , + , and Q fixed to values as determined above, and with − and fitted individually to each of the curves, the measured TRAST curves could be well reproduced ( Figure S11B). Similar to doxyl, the − rate was found to decrease with higher NaAc concentrations ( Figure S11B, insets), consistent with ascorbate donating electrons and acting as a scavenger of hydroxyl radicals, which are needed for the deactivation of the doublet state radicals. Second, we investigated the effects of adding hydrogen peroxide (H2O2), which is formed in the mitochondria upon oxidative phosphorylation. In the TRAST measurements, addition of H2O2 was found to significantly decrease the doublet state population, as opposed to NaAc, but also had no effect on the triplet state population kinetics ( Figure S11C). Having + , and Q fixed to values as determined above, with fitted as a global parameter to the TRAST curves, and − fitted individually to each of the curves, generated fitted curves well in agreement with the measured TRAST curves ( Figure S11C). While the fitted (20.2 ± 2.7 s -1 ) was well in line with previously determined rates, the − rate was found to increase linearly with higher H2O2 concentrations, in contrast to addition of NaAc, but concomitant with the decrease in the doublet state population. As an oxidant and a source of hydroxyl radicals H2O2 here promotes the − rate ( Figure S11C, inset). S10 Figure S1. Comparison -1 refer to the relative relaxed energies of key intermediates between the initial reagents (0 kcal mol -1 ) and products (+51.5 kcal mol -1 ). Figure S6. The fitted + rates from the TRAST measurements in Figure 2D.